By conservative estimates, harmful algal blooms (HABs) cost the United States $50 million per year (Hoagland, P. et al. Estuaries, 2002, 25:819-837). Such estimates are based upon direct economic impacts on tourism, fisheries, etc., and do not account for irremediable costs such as those caused by mass marine mammal mortalities (Landsberg, J. H. Rev. Fish. Sci., 2002, 10:113-390; Landsberg, J. H. and Steidinger, K. A. “A historical review of Gymnodinium breve red tides implicated in mass mortalities of the manatee (Trichechus mantus latirostris) in Florida, USA”, 1998, pp. 97-100, in B. Reguera et al. Eds, Proceedings of the 8th International Conference on Harmful Algae, Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Vigo Spain). Worldwide, algal toxins of all types may be responsible for as many as 60,000 human intoxication events per year (Van Dolah, F. M. et al. Hum. Ecol. Risk Assess., 2001, 7:1329-1345).
Nearly all coastal regions of the United States are impacted by HABs for various intervals in time and intensity. Perhaps no coastal environment has a frequency of HABs equal to that of the Florida Gulf Coast, caused by the nonperidinin dinoflagellate Karenia brevis (Davis) cf. Hansen and Moestrup (=Gymnodinium breve). Although red tides have been observed in the Gulf of Mexico since the Spanish Conquests and reports of catastrophic fish mortalities go back to 1844, the identity of K. brevis, initially named G. breve, as the causative agent was not determined until the bloom of 1946 to 1947 (Gunther, G. et al. Ecol. Monogr, 1948, 18:311-324). In certain years, red tides have occurred during 12 months of the year, although they are most often encountered in the late summer and early fall, correlating with heavy rainfall (Landsberg, J. H. Rev. Fish. Sci., 2002, 10:113-390).
There is a need for monitoring and prediction of HABs, and those of K. brevis are of particular concern. Historically, blooms have occurred primarily during the fall and winter months. However over recent years, the Florida red tide specifically and HABs in general appear to be more prevalent and wide-spread (Chretiennot-Dinet, M., Oceanis, 2001, 24:223-238; Hallegraeff, G. M., Phycologia, 1991, 32:79-99). Massive fish kills, marine mammal mortalities, human poisonings due to the consumption of tainted shellfish and complaints of respiratory irritations among beach-goers are associated with these blooms (Kirkpatrick et al., Harmful Algae, 2004, 3:99-115; Van Dolah et al., in Toxicology of Marine Mammals, Taylor & Francis, Inc., 2002, Vos et al. (Eds.), p. 247-269). These harmful effects are attributed to a suite of polyketide secondary metabolites known as brevetoxins, which are part of a larger family of dinoflagellate-derived polyketide toxins that pose a threat to human health. Brevetoxins are polyether ladder type compounds having two parent backbone structures, brevetoxin A and brevetoxin B, each with several side-chain variants. Examples of other harmful polyketide toxins include ciguatoxin, okadaic acid, and the related kinophysistoxins, pectenotoxins, yessotoxin, and the azaspiracids. The mechanism of synthesis of brevetoxins is unknown but is hypothesized to be the result of enzymes similar to polyketide synthetases. Recently, two polyketide synthetase genes were described from K. brevis (Snyder et al. Mar. Biotechnol., 2003, 5:1-12; Snyder et al. Phytochemistry, 2005, 66(15):1767-80).
A myriad of approaches have been taken to address the problem of HAB monitoring and prediction, including satellite ocean color sensing (Stumpf, R. P. Hum. Ecol. Risk Assess., 2001, 7:1363-1368), photopigment analysis (Millie, D. F. et al. Limnol. Oceanogr., 1997, 42:1240-1251; Millie, D. F. et al. J. Phycol., 2001, 37:35; Oernolfsdottir, E. B. et al. J. Phycol., 2003, 39:449-457), and toxin analysis (Pierce, R. H. and Kirkpatrick, G. J. Environ. Toxicol. Chem., 2001, 20:107-114). Additionally, molecular methods are being developed to detect a variety of HAB species, including Alexandrium sp. (Adachi, M. et al. J. Phycol., 1996, 32:1049-1052; Godhe, A. et al. Mar. Biotechnol., 2001, 3:152-162), Gymnodinium sp. (Godhe, A. et al. Mar. Biotechnol., 2001, 3:152-162; Peperzak, L. et al. “Application and flow cytometric detection of antibody and rRNA probes to Gymnodinium mikimotoi (Dinophyceae) and Pseudo-nitzschia multiseries (Bacillariophyceae), 2000, pp. 206-209, in G. M. Hallegraff et al. Eds., Harmful algal blooms, IOC-UNESCO, Paris, France), Pseudonitzschia sp. (Peperzak, L. et al. “Application and flow cytometric detection of antibody and rRNA probes to Gymnodinium mikimotoi (Dinophyceae) and Pseudo-nitzschia multiseries (Bacillariophyceae), 2000, pp. 206-209, in G. M. Hallegraff et al. Eds., Harmful algal blooms, IOC-UNESCO, Paris, France ), Pfiesteria sp., and Pfiesteria-like organisms (Litaker, R. W. et al. J. Phycol., 2003, 39:754-761) as well as K. brevis (Gray, M. et al. Appl. Environ. Microbiol., 2003, 69:5726-5730; Loret, P. et al. J. Plankton Res., 2002, 24:735-739). All of these methods must be calibrated with microscopy-derived cell counts, which are prone to errors (Culverhouse, P. F. et al. Mar. Ecol. Prog. Ser., 2003, 247:17-25).
Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. Recently, a real-time NASBA assay was developed for the detection of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) large-subunit (rbcL) mRNA from K. brevis (Casper et al., Applied and Environmental Microbiology, 2004, August, 70(8):4727-4732). The rbcL mRNA was selected as the target because cellular levels of mRNA are typically high and RNA degrades quickly in the environment, resulting in detection of viable K. brevis populations only. NASBA RNA amplification occurs at 41° C. (Davey et al., European Patent No. EP 0329822). RNA is amplified by use of an enzyme cocktail including T7 RNA polymerase, avian myeloblastosis virus reverse transcriptase, RNaseH, and two target-specific oligonucleotide primers.
Real-time detection of the amplicon was accomplished by use of a molecular beacon, a single-stranded oligonucleotide that forms a stem-loop structure (Tyagi and Kramer, Nature Biotech., 1996, 14:303-308). The molecular beacon was labeled with 6-carboxy fluorescein (6-FAM) at its 5′ end and quencher DABCYL at its 3′ end. When the beacon is in the closed (hairpin loop) configuration the fluorophore is quenched. Upon binding to the amplicon, the quencher is separated from the fluorophore and the probe fluoresces. During the amplification reactions, the fluorescent signal is measured. The time at which the signal reaches exponential growth is defined as the time to positivity (TTP), which is analogous to the threshold cycle value in PCR. The TTP value is a function of how much initial target RNA is in the sample. This NASBA-based assay for K. brevis rbcL mRNA was used to successfully detect and quantify K. brevis in cultures and field samples collected from the coastal waters of southwest Florida.